磷酸化修饰鬼臼果多糖的制备及生物活性

Phosphorylation modification process, structural characterization, and bioactivity of polysaccharides from the fruits of Podophyllum Emodi

摘要:

以鬼臼果(Podophyllum Emodi)为原料提取鬼臼果多糖(Podophyllum Podophyllum Emodi,PEP),经纯化后采用磷酸钠法制备磷酸化鬼臼果多糖(Phosphorylation Podophyllum Emodi polysaccharides,P-PEP),单因素及响应面法优化PEP磷酸化修饰工艺。综合运用现代光谱、色谱学、X-射线衍射、扫描电镜、热重、核磁(NMR)等手段对修饰前后多糖的理化性质及结构进行表征,并评价修饰前后的自由基清除能力和降血糖活性。研究结果表明磷酸化修饰最佳工艺为:温度72 ℃、pH值9.0、时间5.1 h,此时取代度为11.372%。PEP、P-PEP分子量分别为20 898 Da、21 432 Da,均主要由鼠李糖(Rha)、阿拉伯糖(Ara)、半乳糖(Gal)、葡萄糖(Glu)组成,但修饰前后单糖摩尔比发生显著变化；修饰后多糖结晶度降低、表面形貌发生变化,同时热稳定性提高,在600 ℃时,残碳率分别为21.27%、48.35% 。红外光谱图在1090 cm-1、950 cm-1分别出现P=O、P-O-C磷酸基团特征吸收峰,同时1H NMR 中,δ=5.70 ppm、3.72 ppm处吸收峰以及31P NMR中,2.49 ppm、-6.23 ppm、-7.03 ppm 处吸收峰表明磷酸化修饰成功；PEP、P-PEP对1,1-二甲基-2-三硝基苯肼(DPPH)的IC50分别为0.93 mg/mL、0.60 mg/mL,对2, 2"-联氨-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)的IC50分别为1.05 mg/mL、0.67 mg/mL,对α-葡萄糖苷酶的IC50分别为3.46 mg/mL、1.43 mg/mL；对α-淀粉酶的IC50分别为3.81 mg/mL、0.49 mg/mL,说明磷酸化修饰不仅改善了多糖的理化性质,并且对自由基清除能力和降血糖活性也具有促进作用。

Abstract:

This study extracted polysaccharides from the fruits of PEP. Following purification, phosphoric acid sodium method was employed to synthesize P-PEP. The optimization of the phosphorylation modification process of PEP was conducted using single-factor experiments and response surface methodology. Comprehensive characterization of the physicochemical properties and structures of the polysaccharides before and after modification was performed using modern techniques such as spectroscopy, chromatography, XRD, SEM, thermogravimetric analysis and NMR. Furthermore, the radical scavenging ability and hypoglycemic activity were evaluated for both modified and unmodified polysaccharides. The results indicated that the optimal phosphorylation modification conditions were: temperature 72 °C, pH 9.0, and time 5.1 h, resulting in a degree of substitution of 11.372%. The molecular weights of PEP and P-PEP were determined to be 20 898 Da and 21 432 Da, respectively, both primarily composed of Rha, Ara, Gal, and Glu, with significant changes observed in the molar ratios of monosaccharides before and after modification. The crystallinity of the polysaccharide decreased after modification, and surface morphology changed, while thermal stability improved, with a residual carbon rate of 21.27% and 48.35% at 600 °C, respectively. The infrared spectrum exhibited characteristic absorption peaks of phosphate groups at 1090 cm-1 and 950 cm-1, and 1H NMR spectra displayed absorption peaks at δ=5.70 ppm and 3.72 ppm, while 31P NMR peaks at 2.49 ppm, -6.23 ppm, and -7.03 ppm confirmed successful phosphorylation modification. The IC50 values for PEP and P-PEP against DPPH were 0.93 mg/mL and 0.60 mg/mL, respectively, while against ABTS were 1.05 mg/mL and 0.67 mg/mL, and for α-glucosidase were 3.46 mg/mL and 1.43 mg/mL, respectively. The IC50 values against α-amylase for PEP and P-PEP were 3.81 mg/mL and 0.49 mg/mL, respectively. These findings suggest that phosphorylation modification not only improved the physicochemical properties of the polysaccharides but also enhanced their radical scavenging capacity and hypoglycemic activity.